This revised application proposes studies using cultured rat basal forebrain neurons and ion imaging techniques to investigate mechanisms underlying increases in intracellular [Ca2+] and choline acetyltransferase (ChAT) activity produced by brief exposure to neurotrophins, and neurotrophin-mediated protection from hypoglycemic stress. By imaging neurons filled with the ratiometric Ca-sensitive dye fura2, we have found that certain members of the neurotrophin family, which includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4/5 (NT3, NT4/5), induce transient increases in [Ca2+]i in subsets of basal forebrain neurons. We have also found that brief (15 min) exposure to certain neurotrophins leads to a significant increase in the activity of the acetylcholine-synthesizing enzyme, ChAT, measured 24 hr later. Mechanisms underlying the [Ca2+]i transients will be investigated by determining which neuronal types and neurotrophin receptors are involved, and whether the changes in [Ca2+]i are accompanied by release of internal [Ca2+] stores and/or changes in intracellular [Na+] and/or [H+]. For both the (Ca2+]i and the ChAT responses to brief neurotrophin exposure, we will investigate whether tyrosine (or other) kinase activity is required, whether synaptic or action potential activity is required, and whether the phenomena change during neuronal maturation. We will also determine whether the enhancement of ChAT activity by brief neurotrophin exposure requires changes in [Ca2+]i, transcription, and/or protein synthesis. Our studies have indicated that certain neurotrophins significantly increase basal forebrain neuronal survival and ChAT activity after a hypoglycemic stress. We propose to determine how the degree of protection varies with the identity, concentration and time of application of the neurotrophin. Mechanisms underlying stress protection will be investigated by determining which types of neurons are protected, and whether the stress protection requires tyrosine (or other) kinase activity, transcription and/or protein synthesis, and varies with synaptic activity. These studies will provide important new information concerning how various members of the neurotrophin family exert their trophic effects on basal forebrain neurons, including the central cholinergic neurons implicated in memory mechanisms.